Characterization of Streptococcus pneumoniae isolates obtained from the middle ear fluid of US children, 2011–2021

Introduction Pneumococcal conjugate vaccines (PCVs), including higher valency vaccines such as PCV20, have the potential to reduce pediatric otitis media. We assessed serotype distribution, potential PCV coverage, and antimicrobial susceptibility of Streptococcus pneumoniae isolates cultured from middle ear fluid (MEF) of US children age ≤5 years. Methods S. pneumoniae isolates identified from US hospitals participating in the SENTRY Antimicrobial Surveillance program from 2011 to 2021 were included. Serotypes were determined by in silico analysis based on Pneumococcal Capsular Typing methodology. The percentage of isolates belonging to serotypes included in PCV13 (serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F), PCV15 (PCV13 plus 22F, 33F), and PCV20 (PCV13 plus, 8, 10A, 11A, 12F, 15B, 22F and 33F) was calculated. Antimicrobial susceptibility testing was performed by broth microdilution and interpreted using CLSI criteria. Nonsusceptibility was defined as isolates that were intermediate or resistant to a selected antimicrobial. Results Among the 199 S. pneumoniae isolates that were identified, 56.8% were from children age <2 years. Six serotypes accounted for around 60% of isolates: 35B (16.6%), 15B (14.6%), 15A (7.5%), 19A (7.5%), 19F (7.5%), and 3 (7.0%). Serotypes included in PCV13, PCV15, and PCV20 accounted for 23.1%, 30.2%, and 54.8% of isolates, respectively. Overall, 45.2% of isolates were penicillin non-susceptible, and 13.6% were MDR, of which 48% were serotype 19A. Seven serotypes (19A, 15A, 15B, 15C, 23A, 33F, and 35B) accounted for the majority of non-susceptible isolates. Discussion PCVs, particularly PCV20, may prevent a substantial fraction of S. pneumoniae otitis media (OM), including OM due to non-susceptible serotypes. The addition of serotypes 15A, 23A, and 35B would improve coverage against susceptible and non-susceptible pneumococcal OM.


Introduction
Streptococcus pneumoniae is one of the most common bacterial pathogens causing otitis media (OM).In 2000, a 7-valent pneumococcal conjugate vaccine (PCV7) was licensed and incorporated into the pediatric national immunization program (NIP) in the US, which was replaced by the 13-valent pneumococcal conjugate vaccine (PCV13) in 2010 (1).Among US children born between 2017 and 2018, 82.3% had received four or more doses of PCV13 by age 24 months (2).Since the introduction of pneumococcal conjugate vaccines (PCV) into the pediatric population, there has been a substantial reduction of vaccine serotype OM among children (3)(4)(5)(6).However, despite the success of PCVs, OM disease burden remains substantial (7).Further, a greater proportion of pneumococcal OM cases due to non-PCV13 vaccine serotypes have been reported in the US in recent years (8,9).
The emergence of non-PCV13 vaccine serotypes has led to the development and recommendation of higher-valency PCVs, including a 15-valent PCV (10) and a 20-valent PCV (11).Both vaccines have been approved for use among US children and indicated for prevention of invasive pneumococcal disease (IPD) due to the serotypes contained in the vaccines (10,11).PCV20 is also indicated for prevention of OM caused by the original seven serotypes covered by PCV7 (11).Decisions pertaining to recent recommendations for pediatric PCV use have largely been informed by data from IPD whereas data from OM has been limited (12).While nasopharyngeal samples collected from children with OM have been used to approximate OM serotype distribution (13,14), middle ear fluid (MEF) is still considered the gold standard specimen for detection and characterization of the causative OM agents.
The objective of this study was to assess the pneumococcal serotype distribution and antimicrobial susceptibility including multidrug resistance (MDR), and potential PCV coverage among isolates cultured from MEF obtained from children in the US.

Pneumococcal isolate identification
The SENTRY Antimicrobial Surveillance Program was established in 1997 to monitor pathogens and changes in antimicrobial resistance patterns of organisms collected from patients with various infections (15).Every year, participating laboratories at medical centers in all 9 US Census Divisions identify requested surveillance pathogens by routine microbiologic methods and submit a subculture to the SENTRY program, along with basic demographic information about the case patient and limited information about the clinical setting where the case patient was treated.Pathogen confirmation is conducted by the central laboratory (JMI Laboratories, North Liberty, Iowa) by various techniques including colony morphology, biochemical algorithms, MALDI-TOF MS, PCR assays, and/or sequencing, as needed.From the SENTRY program collection of stored pathogens, S. pneumoniae isolates cultured from MEF samples from children 5 years of age or younger that were submitted from 35 participant laboratories during 2011-2021 were included in this study.Because tympanocentesis is not routinely performed among children with OM in the US and collection technique was not reported, we assumed that the MEF had been collected from a tympanocentesis performed on children with complicated OM (children with recurrent OM, children with a poor response to conventional therapy or children with a clinical relapse) or with a swab from otorrhea from a spontaneously perforated OM (16)(17)(18).We restricted serotyping to S. pneumoniae isolates collected from 2011 through 2021, after introduction of PCV13 into the US pediatric immunization program.

Serotype determination
Serotypes were identified using in silico analysis based on Pneumococcal Capsular Typing methodology (PneumoCaT).Specifically, DNA from S. pneumoniae isolates was extracted using the KingFisher Cell and Tissue DNA kit on a KingFisher TM Flex Magnetic Particle Processor (Thermo Scientific) workstation.Total genomic DNA was used as input material for library construction and sequencing on a NextSeq 1000 Sequencer (Illumina, San Diego, California, USA) using NextSeq TM 1000/2000 P2 Reagents (300 cycles).The DNA libraries for the NextSeq1000 Sequencer were prepared using the Illumina DNA TM library construction protocol and index kit.Rigorous quality control metrics were applied to library construction including verification that the "Quality Score" (% ≥Q30) for the run was above 75%, the percentage of clusters passing filter was ≥60%, and the loading concentration was ≥95.Each raw data set was de novo assembled using SPAdes 3.11.1.PneumoCaT (v.1.2.1) used a two-step approach to assign capsular type to S. pneumoniae genomic data.In the first step, if the reads matched >90% to one or more of the 92 serotypes for S. pneumoniae plus 2 additional subtypes/molecular types, a capsular type was assigned.If more than one loci matched, then in the second step, a variant-based approach that utilizes the capsular type variant database to distinguish serotypes within a subgroup/genogroup was applied to make a call on the serotype.If the coverage value against the reference sequence was ≤90%, then the serotype analysis reported "Failed" as its value.Average depth of coverage across the matching region of ≥30X was considered acceptable for PneumoCaT to analyze the data (19,20).The nucleotide sequences used to assign a serotype were submitted to the National Center for Biotechnology Information (NCBI) and were assigned accession numbers SAMN41612263 -SAMN41612461.

Susceptibility testing
Minimum Inhibitory Concentrations (MICs) were determined by the broth microdilution method using Clinical and Laboratory Standards Institute (CLSI) guidelines (2018) (21).Reference broth microdilution panels were manufactured at JMI Laboratories (2015-2021) or purchased from Thermo Fisher Scientific (2011-2014) (Cleveland, Ohio, USA) using freshly prepared drug stocks and stored at −80°C until use.Testing was performed in cationadjusted Mueller-Hinton broth supplemented with 2.5% to 5.0% lysed horse blood (MHB-LHB).Antimicrobial susceptibility testing was performed for amoxicillin-clavulanic acid, penicillin, ceftriaxone, clindamycin, erythromycin, trimethoprim/ sulfamethoxazole (TMP/SMX), levofloxacin, and vancomycin.MIC results were interpreted as susceptible, intermediate, or resistant according to the CLSI recommendations (22).Nonmeningitis breakpoints were used for penicillin and ceftriaxone.Non-susceptible isolates were defined as those isolates that were intermediate or resistant to a selected antimicrobial agent.Multidrug resistance (MDR) was defined as resistant to three or more classes of antimicrobials.The MIC 50 was defined as the MIC of a given antimicrobial drug that inhibited growth of 50% of isolates, and MIC 90 was defined as the MIC that inhibited growth of 90% of the isolates.

Study population
A total of 199 S. pneumoniae isolates from middle ear fluid among children 5 years of age or younger were collected by the SENTRY program from 2011 to 2021.All 199 S. pneumoniae were serotyped (by sequencing) and tested for antimicrobial susceptibility (Table 1).Of these, 113 isolates (56.8%) were from children <2 years of age.Most isolates were collected from children receiving care in the ear, nose, and throat (ENT; n = 89, 44.7%) or pediatrics (n = 61, 30.7%) departments.Approximately 60% of isolates came from male children, and about 50% were isolated between 2017 and 2021.While isolates were reported from all 9 US Census regions, most (57.3%)originated from the West North Central region.

Potential vaccine serotype coverage
In children aged ≤5 years, PCV13, PCV15, and PCV20 serotypes accounted for 23.1%, 30.2%, and 54.8% of S. pneumoniae isolates, respectively and 45.2% of cases were due to Non-PCV20 serotypes (Table 2).The PCV20 non-PCV13 serotypes and PCV15 non-PCV13 serotypes contributed 31.7% and 7.5% of cases, respectively (Table 2).Considering the age group, while PCV20 coverage was similar for children in both age groups, PCV20 non-PCV13 coverage was higher for children <2 years than in children 2-5 years of age (37.2% vs. 20.9%,respectively; p-value = 0.01) and this was due to a larger percentage of serotype 15B isolates detected in the younger age group.Overall, the percentage of serotypes included in PCV20 was higher in P2 than P1 (57.7% vs. 48.0%,respectively; p-value = 0.17) as was also true for PCV20 non-PCV13 serotypes (35.1% vs. 25.5%,respectively; p-value = 0.14) (Table 2).Serotype distribution by age group and study period are presented in Supplementary Table S2.

Discussion
In this study we assessed the serotype distribution, potential coverage by available pneumococcal vaccines, and antimicrobial susceptibility for pneumococcal isolates cultured from the MEF of US children with OM.Our results showed that more than half of S. pneumoniae isolates were serotypes covered by PCV20 (52.8%), 30.2% were covered by PCV15, and 22.6% were covered by PCV13.Serotypes 35B (16.6%) and 15B (14.6%) were the most common serotypes overall followed by serotypes 15A, 3, 19A, and 19F, each accounting for approximately 7% of cases.Together, these serotypes accounted for nearly two-thirds (61%) of pneumococcal isolates identified from MEF samples.
Other studies from US and European children that cultured and serotyped S. pneumoniae from MEF also reported that these serotypes were commonly identified.In these studies, and ours, the most common PCV13 serotypes collected from MEF were serotypes 3, 19A, and 19F (27-30).In the two US studies conducted during the PCV13 period among children with intact or perforated OM, serotypes 15A, 15B/C, 23B, and 35B also were among the most commonly identified (27,29).In German and French children, serotypes 15B/C, 23B, and 35B, but not 15A as in our study, were among the most common serotypes detected in MEF.In addition, serotypes 10A, 11A, 16F, 24F were also common among French children only (28,30).Several explanations could account for these aggregate results.For PCV13 serotypes 3, 19A, and 19F, pre-licensure immunogenicity studies demonstrated that PCV13 elicited a robust immune response and functional antibody activity for each of these serotypes, particularly after the booster dose (31).Post-licensure studies reported that following PCV introduction     (32), while PCV13 protected against each of these three serotypes in Israel (3).These data suggest that residual disease due to these serotypes could be attributed to lower effectiveness against carriage, including potentially against carriage acquisition, density, or duration of the immune response (33)(34)(35)(36).For example, in our study, serotype 3 was more common among children 2-5 years (12.8%)than children <2 years of age (2.7%), which could reflect a shorter duration of immunity for serotype 3 (37).Alternatively, residual disease due to these serotypes could also be due to a lower amount of antibody present in the MEF or with a capsular phenotype of some serotypes (e.g., serotype 3) that facilitates progression to disease (38).
For the most common non-PCV13 serotypes identified in this study (15A, 15B, 15C, 23B, 35B), not only have they become increasingly common colonizers over subsequent eras of PCV use, but they have also been frequently associated with antimicrobial nonsusceptibility, especially serotypes 15A and 35B (9,27,39).Serotype 35B has also become both a more common invasive serotype and acquired multiclass non-susceptibility among US children with IPD (40).The emergence of these nonsusceptible strains likely results from selection pressure created by exposure to antimicrobials (41).Strains possessing both nonsusceptibility and virulence genes may become more common causes of systemic and mucosal disease as was observed among certain non-vaccine serotypes after PCV7 introduction (40,42).
Overall, a substantial proportion of isolates in each PCV serotype group was non-susceptible to at least one antimicrobial.While non-susceptibility was seen for at least one isolate for most of the 23 serotypes identified, most non-susceptible isolates were accounted for by 7 serotypes: 19A, 15A, 15B, 15C, 23A, 33F, and 35B, most of which were also reported by other studies (9,40).We also observed a decrease in the proportion of nonsusceptible and MDR PCV13 serotypes over the study time periods, primarily driven by the decrease of serotype 19A, as has been reported previously (40,43).PCV15 includes serotypes 19A and 33F and PCV20 also includes serotypes 15B and possibly 15C through cross protection (24), with the latter potentially covering 39% of penicillin non-susceptible isolates.Serotypes 15A, 23A, and 35B are not currently covered by any higher valency PCVs, and while disease incidence for these serotypes has not increased, non-susceptibility to antimicrobials among these serotypes is common, which makes them important candidates to include in future higher valency PCVs (40).Importantly, serotype inclusion and PCV coverage estimates may not linearly translate to protection due to immune interference and the possible need for higher levels of antibody to prevent mucosal disease (36).
Our study had limitations.Because the source of many S. pneumoniae isolates was presumably from children with spontaneously perforated OM, the serotype distribution of these cases may not be representative of all OM cases.Specifically, the majority of children were seen in the ENT or pediatrics departments which could be indicative of prior antibiotic treatment failure.Clinical data such as previous use of antimicrobials and vaccine history were not available, and therefore, it was not possible to associate S. pneumoniae isolation with therapeutic failure or vaccine breakthrough infection.Also, the SENTRY program is a laboratory-based surveillance system designed to observe distribution of pathogens and antimicrobial resistance patterns for any given infection based on a prespecified target number of pathogens per year.Therefore, the true prevalence of S. pneumoniae serotypes cannot be ascertained with these sampling criteria.However, this study demonstrates that antimicrobial resistance surveillance systems can be leveraged to understand serotype distribution of pneumococcal isolates from MEF samples.
Our study showed that certain PCV13 serotypes -3, 19A, and 19Fare frequently isolated from MEF of children even in the context of a mature pediatric PCV13 program, emphasizing the need for continued monitoring following the introduction of higher valency vaccines such as PCV15 and PCV20.Furthermore, among the pneumococcal isolates in this study, an important fraction were attributed to serotypes beyond those in PCV13 that are covered by higher valency PCVs.Among the additional serotypes in PCV20 beyond PCV13, several were associated with antimicrobial non-susceptibility and MDR.Therefore, including PCV20 in existing pediatric national immunization programs may further reduce the frequency of overall and antimicrobial non-susceptible S. pneumoniae.With the introduction of higher valency PCVs, further studies are needed to monitor the changes in pneumococcal epidemiology in children with OM in the long term.Future PCVs should consider including non-PCV20 serotypes, particularly 15A, 23A, and 35B, to further reduce the burden of OM among children.

TABLE 1
Characteristics of S. pneumoniae isolated from MEF samples.

TABLE 4
Antimicrobial nonsusceptibility of S. pneumoniae serotypes isolated from MEF from children ≤5 years, 2011-2021.Results for Levofloxacin and Vancomycin are not included because all isolates were susceptible to these antimicrobials.No nonsusceptible isolates were identified for b Nonsusceptible is defined as intermediate or resistant.c